Download The role of tumor necrosis factor-alpha

170
Research Article
The role of tumor necrosis factor-alpha -308
G/A and transforming growth factor-beta 1 -915
G/C polymorphisms in childhood idiopathic
thrombocytopenic purpura
Çocukluk çağı idiopatik trombositopenik purpura'da TNF-α-308G/A ve
TGF-β1-915G/C polimorfizmlerinin rolü
Emel Okulu1, Talia İleri2, Vildan Koşan Çulha3, Fatih Mehmet Azık3, Yonca Eğin4,
Zümrüt Uysal2, Nejat Akar4
1Department
of Pediatrics, Faculty of Medicine, Ankara University, Ankara, Turkey
2Department of Pediatrics, Division of Pediatric Hematology, Faculty of Medicine, Ankara University, Ankara, Turkey
3Department
of Pediatric Hematology, Ankara Dışkapı Children’s Health Training and Research Hospital,
Ankara, Turkey
4Department of Pediatrics, Division of Pediatric Molecular Pathology and Genetics, Faculty of Medicine, Ankara
University, Ankara, Turkey
Abstract
Objective: To increase our understanding of the etiology of idiopathic thrombocytopenic purpura (ITP)
some cytokine gene polymorphisms were analyzed for susceptibility to the disease. The aim of this
study was to investigate the role of tumor necrosis factor-alpha (TNF-α) -308 G/A and transforming
growth factor-beta 1 (TGF-β1) –915 G/C polymorphisms in the development and clinical progression
of childhood ITP.
Materials and Methods: In all, 50 pediatric patients with ITP (25 with acute ITP and 25 with chronic
ITP) and 48 healthy controls were investigated via LightCycler® PCR analysis for TNF-α -308 G/A and
TGF-β1 -915 G/C polymorphisms.
Results: The frequency of TNF-α -308 G/A polymorphism was 20%, 16%, and 22.9% in the acute ITP
patients, chronic ITP patients, and controls, respectively (p>0.05). The frequency of TGF-β1 -915 G/C
polymorphism was 16%, 8%, and 8.3% in the acute ITP patients, chronic ITP patients, and controls,
respectively (p>0.05). The risk of developing ITP and clinical progression were not associated with
TNF-α -308 G/A (OR: 0.738, 95% CI: 0.275-1.981, and OR: 0.762, 95% CI: 0.179-3.249) or TGF-β1 -915
G/C (OR: 1.5, 95% CI: 0.396-5.685, and OR: 0.457, 95% CI: 0.076-2.755) polymorphisms.
Conclusion: The frequency of TNF-α -308 G/A and TGF-β1 -915 G/C polymorphisms did not differ
between pediatric ITP patients and healthy controls, and these polymorphisms were not associated with
susceptibility to the development and clinical progression of the disease. (Turk J Hematol 2011; 28: 170-5)
Key words: Idiopathic thrombocytopenic purpura, childhood, TNF-α, TGF-β1, polymorphism
Received: May 24, 2010
Accepted: December 14, 2010
Address for Correspondence: M.D. Emel Okulu, Department of Pediatrics, Faculty of Medicine, Ankara University, 06100 Dikimevi, Ankara,
Turkey Phone: +90 312 595 63 90 E-mail: [email protected]
doi:10.5152/tjh.2011.50
Okulu et al.
Polymorphisms in childhood ITP
Turk J Hematol 2011; 28: 170-5
171
Özet
Amaç: İdiopatik trombositopenik purpura (ITP)’nın etyolojisini anlayabilmek için, hastalığa yatkınlık
üzerinde bazı sitokin gen polimorfizmleri incelenmiştir. Bu çalışmanın amacı çocukluk çağı ITP’sinin
gelişimi ve klinik seyrinde tümör nekrozis faktör-alfa (TNF-α) –308G/A ve transforming growth faktörbeta1 (TGF-β1) -915G/C polimorfizmlerinin rolünü araştırmaktır.
Yöntem ve Gereçler: Elli ITP’li çocuk hasta (25 akut ITP, 25 kronik ITP) ve 48 sağlıklı kontrol TNF-α–
308G/A ve TGF-β1–915G/C polimorfizmleri için Light Cycler PCR analizi ile araştırıldı.
Bulgular: Akut ITP, kronik ITP ve kontrollerde TNF-α–308G/A polimorfizm sıklığı sırasıyla %20, %16 ve
%22.9 (p>0.05), ve TGF-β1–915G/C polimorfizm sıklığı sırasıyla %16, %8 ve %8.3 (p>0.05) idi. ITP
gelişim riski ve klinik seyri ile TNF-α–308G/A (OR: 0.738, %95 Cl: 0.275-1.981 ve OR: 0.762, %95 Cl:
0.179-3.249) ve TGF-β1–915G/C (OR: 1.5, %95 Cl: 0.396-5.685 ve OR: 0.457, %95 Cl: 0.076-2.755)
polimorfizmleri arasında ilişki bulunamadı.
Sonuç: Bu sonuçlar, çocukluk çağı ITP hastalarında TNF-α–308G/A ve TGF-β1–915G/C polimorfizmlerinin sıklığının sağlıklılardan farklı olmadığını göstermiş olup, bu polimorfizmlerin ITP gelişimi ve hastalığın klinik seyri için risk oluşturmadığını düşündürmektedir. (Turk J Hematol 2011; 28: 170-5)
Anahtar kelimeler: İdiopatik trombositopenik purpura, çocukluk çağı, TNF-α, TGF-β1, polimorfizm
Geliş tarihi: 24 Mayıs 2010
Kabul tarihi: 14 Aralık 2010
Introduction
Idiopathic thrombocytopenic purpura (ITP) is
one of the most frequent causes of acquired thrombocytopenia and is characterized by destruction of
autoantibody-mediated platelets. The pathophysiology of ITP is complex and includes antibodies, cytokines, antigen-presenting cells, co-stimulatory molecules, and T- and B-lymphocytes. When autoreactive B-lymphocytes produce anti-platelet antibodies,
T-lymphocytes play an important role in this autoimmune process [1-3].
Children with ITP have a Th1-type cytokine pattern with elevated levels of tumor necrosis factoralpha (TNF-α), as in most autoimmune diseases [4].
Researches have shown that polymorphisms in the
TNF-α gene at position -308 and -238 affect gene transcription with increased TNF-α production [5,6]. The
transcriptionally more active allele of TNF-α (allele 2
of -308) was less frequently observed in pediatric
chronic ITP patients than in healthy controls [7].
Transforming growth factor-beta 1 (TGF-β1) is
another multifunctional cytokine that plays a critical
role in the pathogenesis of inflammatory, autoimmune, and malignant diseases. The expression of
TGF-β1 is influenced by polymorphisms in the TGFβ1 gene, which may be partly relevant to certain diseases [8-10]. It was reported that some TGF-β1 polymorphisms are not associated with the development
and progress of ITP [11]. The aim of the present
study was to determine if TNF-α -308 G/A and TGF-β1
-915 G/C polymorphisms play a role in the development and clinical progression of ITP in children.
Materials and Methods
Study population
The study was conducted at Ankara University,
Faculty of Medicine, Department of Pediatric
Hematology, Pediatric Molecular Pathology and
Genetics Division, and at Ankara Dışkapı Children’s
Health Training and Research Hospital, Department
of Pediatric Hematology. In all, 50 pediatric ITP
patients (25 acute ITP and 25 chronic ITP) and 48
healthy controls were enrolled in this study. Children
diagnosed with acute or chronic ITP and regularly
followed-up in the hospitals participating in the
study were considered for inclusion.
The diagnosis of ITP was based on medical history,
physical examination, and thrombocytopenia.
Thrombocytopenia that resolved within 6 months of
onset was defined as acute ITP and thrombocytopenia that persisted for >6 months was defined as
chronic ITP. All the controls had a negative history of
thrombocytopenia and ITP, and their blood samples
were obtained from the DNA bank of Ankara University,
Faculty of Medicine, Department of Pediatric
Hematology, Pediatric Molecular Pathology and
Genetics Department. Informed consent was obtained
from all the patients, the controls, and their parents.
The study protocol was approved by the Ankara
University Faculty of Medicine Ethics Committee.
Genomic DNA analysis, and TNF-α -308 G/A and
TGF-β1 -915 G/C polymorphism genotyping
Peripheral blood samples were obtained from all
the participants. Blood samples were collected in
172
Okulu et al.
Polymorphisms in childhood ITP
Turk J Hematol 2011; 28: 170-5
microfuge tubes containing ethylenediamine tetraacetic acid as anticoagulant. Genomic DNA was
extracted from peripheral blood leukocytes using
the phenol-chloroform method.
TNF-α -308 polymorphism
Regions containing TNF-α -308 G/A polymorphisms were amplified via polymerase chain reaction (PCR) using 0.1 μg/mL concentration of 2
primer sets, according to our previous report [12].
TGF-β1 -915 polymorphism
Regions containing TGF-β1 -915 G/C polymorphisms were amplified via PCR using 0.1 μg/mL
concentration of 2 primer sets. The primer sequences used for the TGF-β1 -915 promoter region were
as follows:
Forward: 5’-ACC ACA CCA GCC CTG TTC GC-3’;
Reverse: 5’-AGC TTG GAC AGG ATC TTG CC-3’.
PCR yielded a 224-base pair (bp) long oligonucleotide product and the results were confirmed
based on 2% agarose gel electrophoresis containing 0.5 mg/mL ethidium bromide at 100 W for 45
min using an 18-μL PCR product. The gels were
evaluated with a UV transilluminator imaging analyzer.
Statistical analysis
Statistical analysis was performed using SPSS
v.15.0 (Statistical Package for Social Sciences, Inc.,
Chicago, IL, USA). The demographic features of
the acute and chronic ITP patients were compared
using Student’s t-test and the Mann-Whitney U-test.
The odds ratio and 95% confidence interval (CI),
as estimates of the relative risk of allele frequency,
were calculated for the entire study population.
The 95% CI was calculated based on a conditional
logistic regression algorithm using the maximum
likelihood method.
Results
In the present study 50 ITP patients (25 acute ITP
and 25 chronic ITP) and 48 controls were analyzed
for TNF-α -308 G/A and TGF-β1 -915 G/C polymorphisms. The demographics of the acute and chronic
ITP patients are shown in Table 1. The acute ITP
patients were aged 4 months to 12 years (median
age: 6 years), and the chronic ITP patients were
aged 18 months to 15.5 years (median age: 9 years).
The patients with chronic ITP were significantly
older (p<0.05). Hemoglobin, leukocyte, and platelet counts in the acute and chronic ITP patients at
admission did not differ significantly (p=0.12, 0.99,
and 0.64, respectively).
In total, 5 (20%) of the 25 acute ITP patients, 4
(16%) of the 25 chronic ITP patients, and 11 (22.9%)
of the 48 control subjects had TNF-α -308 G/A gene
polymorphism. The frequency of TNF-α -308 G/A
gene polymorphism in the acute ITP, chronic ITP,
and control groups did not differ significantly
(p>0.05). In addition, the A allele frequency did not
differ significantly between the 3 groups (p>0.05).
The frequency of TGF-β1 -915 G/C polymorphism
in the acute ITP, chronic ITP, and control groups was
16%, 8%, and 8.3%, respectively, but the difference
was not significant (p>0.05). C allele frequency was
2-fold higher in the acute ITP patients (8%) than in
the chronic ITP patients (4.1%) and controls (4%);
the difference was not statistically significant
(p>0.05). The distribution of TNF-α -308 G/A and
TGF-β1 -915 G/C polymorphisms in the study and
control groups are shown in Tables 2 and 3.
The risk of developing ITP was not associated
with TNF-α -308 G/A (OR: 0.738, 95% CI: 0.275-1.981)
or TGF-β1 -915 G/C (OR: 1.5, 95% CI: 0.396-5.685)
polymorphisms. The frequency of TNF-α -308 G/A
and TGF-β1 -915 G/C polymorphisms in the acute
and chronic ITP patients did not differ significantly
Table 1. Patient demographics
Age (years)
Sex (M/F)
Hemoglobin (g/dl)*
Leukocyte
Platelet
(/mm3)*
(/mm3)*
*Results are expresed as median (range)
Acute ITP (n=25)
Chronic ITP
(n=25)
p
5.6±3.5
8.0±3.9
0.028
15/10
11/14
>0.05
11.5 (8.0-14,9)
11.9 (6.8-14.5)
>0.05
8400 (4900-17300)
8100 (5000-23900)
>0.05
8000 (1000-25000)
8000 (1000-78000)
>0.05
Okulu et al.
Polymorphisms in childhood ITP
Turk J Hematol 2011; 28: 170-5
173
Table 2. Distribution of TNF-α-308G/A polymorphism in ITP, acute ITP, chronic ITP patients and controls
Genotype frequency
Controls
n=48
n (%)
ITP patients
n=50
n(%)
Acute ITP
n=25
n (%)
Chronic ITP
Fischer’s
(n=25)
exact test
n (%)
OR (95% CI)
G/G
37 (77.1)
41 (82)
20 (80)
21 (84)
0.546a
0.738 (0.275-1.981)
G/A
11 (22.9)
9 (18)
5 (20)
4 (16)
1.000b
0.762 (0.179-3.249)
OR Odds ratio; CI confidence interval, aITP group compared with the controls, bAcute ITP group compared with chronic ITP group
Table 3. Distribution of TGF-β1-915G/C polymorphism in ITP, acute ITP, chronic ITP patients and controls
Genotype
frequency
Controls
n=48
n (%)
ITP patients
n=50
n (%)
Acute ITP
n=25
n (%)
Chronic ITP
Fischer’s
n=25
exact test
n (%)
OR (95% CI)
G/G
44 (91.7)
44 (88)
21 (84)
23 (92)
0.741a
1.5 (0.396-5.685)
G/C
4 (8.3)
6 (12)
4 (16)
2 (8)
0.667b
0.457 (0.076-2.755)
OR Odds ratio; CI confidence interval, aITP group compared with the controls, bAcute ITP group compared with chronic ITP group
(p>0.05), and these polymorphisms did not have an
affect on chronic progression of the disease
(OR: 0.762, 95% CI: 0.179-3.249 and OR: 0.457, 95%
CI: 0.076-2.755, respectively).
Discussion
Although the etiology of ITP remains unclear, it is
generally accepted that both environmental and
genetic factors play an important role in the development of the disease [13,14]. Many recent studies
have focused on the association between some
cytokine gene polymorphisms and susceptibility to
ITP. Studies that investigated genetic risk factors in
the pathogenesis of ITP reported that polymorphisms of HLA class II antigens, FcγRIIA and
FcγRIIIA, and human platelet antigen (HPA) systems
were associated with the development of ITP and
response to treatment [7,15-19].
TNF-α is a pleiotropic cytokine produced primarily by macrophages and T-cells, and has a range of
inflammatory and immunomodulatory activity
[20-22]. Polymorphisms of TNF-α promoter are associated with high levels of TNF-α and have been studied as a determinant of susceptibility to numerous
diseases [12,21-25]. A study that examined inflammatory cytokines and Fcγ receptor polymorphisms in
chronic childhood ITP reported that polymorphisms
in 2 low-affinity FcγR genes (IIIA and IIIB) and 2 proinflammatory cytokine genes (TNF and LTA) were
associated with chronic childhood ITP [7]. To the
best of our knowledge the role of TNF-α -308 G/A
polymorphism in the development and progression
of ITP has not been previously studied.
TGF-β is a multifunctional cytokine and TGF-β1 is
the most abundant isoform [26,27]. TGF-β1 gene
polymorphisms that alter TGF-β1 production have
been studied in predicting susceptibility to certain
diseases [8,9]. A significant increase in the percentage of NK cells and elevated TGF-β1 levels have
been observed in chronic ITP patients in remission
[28]. Although it has been shown that TGF-β1 gene
polymorphisms (509 G/C, codon 25, and codon 10)
were significantly correlated with many diseases
and TGF-β1 production, the only study on the role of
these polymorphisms in the pathophysiology of ITP
reported that they weren’t correlated with the
development, clinical progression, or treatment
response of the disease [11,29-32].
In the present study the genotype frequency of
TNF-α -308 G/A and TGF-β1 -915 G/C polymorphisms
was evaluated in terms of their correlation with susceptibility to the development and progression of
ITP. At the time of diagnosis it is not possible to predict the progress of the disease. To date, no prognostic factors for the development and clinical progression of the disease have been reported [33].
The results of the present study show that the frequency of TNF-α -308 G/A and TGF-β1 -915 G/C polymorphisms in the acute ITP, chronic ITP, and control
groups did not differ significantly. Moreover, associations between these polymorphisms, and the
development and clinical progress of ITP were not
observed.
Plasma TNF-α and TGF-β1 levels in the patient
and control groups were not analyzed in the present
study. It is known that a substitution at the -308 position of TNF-α results in increased production of
174
Okulu et al.
Polymorphisms in childhood ITP
TNF-α [6], and that polymorphisms at codon 10 and
25 of the TGF-β1 gene are involved in the upregulation of TGF-β1 secretion. Other studies failed to
observe any association between serum TGF-β1
levels and these polymorphisms [34].
In conclusion, TNF-α -308 G/A and TGF-β1 -915
G/C polymorphisms did not play an important role as
genetic risk factors for the development and progression of ITP. As the study and control groups were
small, different results may be obtained by additional
research involving larger patient populations.
Turk J Hematol 2011; 28: 170-5
9.
10.
11.
12.
13.
Acknowledgement
This study was supported by the Ankara University
Research Fund (project number 20030809168).
Conflict of interest statement
The authors of this paper have no conflicts of
interest, including specific financial interests, relationships, and/or affiliations relevant to the subject
matter or materials included.
References
1.
2.
3.
4.
5.
6.
7.
8.
Nugent DJ. Childhood immune thrombocytopenic purpura. Blood Reviews 2002;16:27-9.
Blanchette V, Bolton-Maggs P. Childhood immune
thrombocytopenic purpura: Diagnosis and management. Hematol Oncol Clin N Am 2010;24:249-73.
Yaprak I, Atabay B, Durak I, Turker M, Oniz H, Ozer EA.
Variant clinical courses in children with immune thrombocytopenic purpura: Sixteen year experience of a single medical center. Turk J Hematol 2010;27:147-55.
Andersson J. Cytokines in idiopathic thrombocytopenic purpura (ITP). Acta Paediatr Suppl 1998;424:61-4.
Bidwell JL, Wood NA, Morse HR, Olomolaiye OO, Keen
LJ, Laundy GJ. Human cytokine gene nucleotide
sequence aligments: supplement 1. Eur J Immunogenet
1999;26:135-223.
Wilson AG, Symons JA, McDowell TL, McDevitt HO,
Duff GW. Effects of a polymorphism in the human
tumor necrosis factor α promoter on transcriptional
activation. Proc Natl Acad Sci USA 1997;94:3195-9.
Foster CB, Zhu S, Erichsen HC, Lehrnbecher T, Hart ES,
Choi E, Stein S, Smith MW, Steinberg SM, Imbach P,
Künhe T, Chanock SJ; Early Chronic ITP Study Group.
Polymorphisms in inflammatory cytokines and Fcγ receptors in childhood chronic immune thrombocytopenic
purpura: a pilot study. Br J Haematol 2001;113:596-9.
Chang WW, Su H, He L, Zhao KF, Wu JL, Xu ZW.
Association between transforming growth factor-β1
T869C polymorphism and rheumatoid arthritis: a metaanalysis. Rheumatology 2010;49:652-6.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
Blobe GC, Schieman WP, Lodish HF. Role of transforming growth factor-β in human disease. N Eng J Med
2000;18:1350-8.
Letterio JJ, Roberts AB. Regulation of immune responses by TGF-β. Annu Rev Immunol 1998;16:137-61.
Atabay B, Oren H, Irken G, Kizildağ S, Tunali S, Türker
M, Yilmaz S. Role of transforming growth factor-β1
gene polymorphisms in childhood idiopathic thrombocytopenic purpura. J Pediatr Hematol Oncol
2003;25:885-9.
Akar N, Hasipek M. Tumour necrosis factor-alpha gene
polymorphism (-308 G-A) in Turkish pediatric thrombosis patients. Turk J Hematol 2002;19:39-41.
George JN, Woolf SH, Raskob GE, Wasser JS, Aledort
LM, Ballem PJ, Blanchette VS, Bussel JB, Cines DB,
Kelton JG, Lichtin AE, McMillan R, Okerbloom JA,
Regan DH, Warrier I. Idıopathic thrombocytopenic purpura: A practice guideline developed by explicit methods fort he American Society of Hematology. Blood
1996;88:3-40.
Richardson B. Primer; Epigenetics of autoimmunity.
Nat Clin Pract Rheumatol 2007;3:521-7.
Castro V, Oliveira GB, Origa AF, Annichino-Bizzacchi
JM, Arruda VR. The human platelet alloantigen 5 polymorphism as a risk fort he development of acute idiopathic purpura. Thromb Haemost 2000;84:360-1.
Thude H, Gatzka E, Anders O, Barz D. Allele frequencies of human platelet antigen 1,2,3 and in normal
people. Vox Sang 1999;77:149-53.
Karpatkin S, Fotino M, Gibofsky A, Winchester RJ.
Association of HLA-Drw2 with autoimmune thrombocytopenic purpura. J Clin Invest 1979;63:1085-8.
Fujimoto TT, Inoue M, Shimomura T, Fijumura K.
Involvement of Fcγ receptor polymorphism in the
therapeutic response of idiopathic thrombocytopenic
purpura. Br J Haematol 2001;115:125-30.
Cines DB, Blanchette VS. Immune thrombocytopenic
purpura. N Eng J Med 2002;346:995-1008.
Allen RD. Polymorphism of the human TNF-alpha promoter-random variation or functional diversity? Mol
Immunol 1999;36:1017-27.
Hajeer AH, Hutchinson IV. Influence of TNF-alpha gene
polymorphisms on TNF alpha production and disease.
Hum Immunol 2001;62:1191-9.
Hajeer AH, Hutchinson IV. TNF-alpha gene polymorphism: clinical and biological implications. Microsc
Res Tech 2000;50:216-28.
Tekeli O, Turacli ME, Egin Y, Akar N, Elhan AH. Tumor
necrosis factor alpha-308 gene polymorphism and pseudoexfoliation glaucoma. Mol Vision 2008;14:1815-8.
Wilson AG, Symons JA, McDowell TL, McDevitt HO,
Duff GW. Effects of polymorphism in the human tumor
necrosis factor α promoter on transcriptional activation. Proc Natl Acad Sci USA 1997;94:3195-9.
Batikhan H, Gokcan MK, Beder E, Akar N, Ozturk A,
Gerceker M. Association of the tumor necrosis factoralpha -308 G/A polymorphism with nasal polyposis. Eur
Arch Otorhinolaryngol 2010;267:903-8.
Turk J Hematol 2011; 28: 170-5
26. Massague J. Transforming growth factor-alpha. A
model for membrane-anchored growth factors J Biol
Chem 1990;265:21393-6.
27. Lyons RM, Gentry LE, Purchio AF, Moses HL. Mechanism
of activation of latent recombinant transforming
growth factor beta 1 by plasmin. J Cell Biol
1990;110:1361-7.
28. Andersson PO, Stockelberg D, Jacobsson S, Wadenvik
V. A transforming growth factor β1-mediated bystander
immune suppression could be associated with remission of chronic idiopathic thrombocytopenic purpura.
Ann Hematol 2000;79:507-13.
29. Grainger DJ, Heathcote K, Chiano M, Snieder H, Kemp
PR, Metcalfe JC, Carter ND, Spector TD. Genetic control of the circulating concentration of transforming
transforming growth factor type β1. Hum Mol Genet
1999;8:93-7.
30. Awad MR, El-Gamel A, Hasleton P, Turner DM, Sinnott
PJ, Hutchinson IV. Genotypic variation in the transforming growth factor-β1 gene: association with transforming growth factor-β1 production, fibrotic lung disease and graft fibrosis after lung transplantation.
Transplantation 1998;66:1014-20.
Okulu et al.
Polymorphisms in childhood ITP
175
31. Yamada Y, Miyauchi A, Takagi Y, Tanaka M, Mizuno M,
Harada A. Association of the C509→T polymorphism,
alone or in combination with the T869→C polymorphism, of the transforming growth factor-β1 gene with
bone mineral density and genetic susceptibility to
osteoporosis in Japanese women. J Mol Med
2001;79:149-56.
32. Niu W, Qi Y, Gao P, Zhu D. Association of TGFB1 -509
C>T polymorphism with breast cancer: evidence from
a meta-analysis involving 23,579 subjects. Breast
Cancer Res Treat 2010;124:243-9.
33. Jayabose S, Levendoglu-Tugal O, Ozkaynak MF,
Visintainer P, Sandoval C. Long-term outcome of
chronic idiopathic thrombocytopenic purpura in children. J Pediatr Hematol Oncol 2004;26:724-6.
34. Hinke V, Seck T, Clanget C, Scheidt-Nave C, Ziegler R,
Pfeilschifter J. Association of transforming growth factor-beta1 (TGFbeta1) T29 –4 C gene polymorphism
with bone mineral density (BMD), changes in BMD,
and serum concentrations of TGF-beta1 in a population-based sample of postmenopausal German
women. Calcif Tissue Int 2001;69:315–20.